Process for cultivating acetic acid-containing yeasts

ABSTRACT

A process for cultivating an acetic acid-consuming yeast includes the following steps: 1. An acetic acid-consuming yeast is added to a growthsupporting mixture of aqueous nutrient medium, acetic acid at a low concentration level and gas containing free oxygen. 2. Aerobic culturing of the yeast is initiated with no noticeable stationary growth stage by incrementally introducing small amounts of acetic acid into the fermentation mass either continuously or intermittently throughout the growth period of the micro-organism.

United States Patent [191 Kinsel et a1. A l

[ 1 Nov. 27, 1973 PROCESS FOR CULTIVATING ACETIC ACID-CONTAINING YEASTS[75] Inventors: Norma A.'Kinsel, Pittsburgh;

William W. Leathen, Wexford, both of Pa.

[73] Assignee: Gulf Research & Development Company, Pittsburgh, Pa.

[22] Filed: Sept. 17, 1971 [21] Appl. No.: 181,557

OTHER PUBLICATIONS Ogata et al., Agr. Biol. Chem, Vol. 33, pp. 977-978,

1969. Ogata et al., Chem. Abs., Vol. 73, No. 119l79n, 1970.

Primary Examiner-Alvin E. Tanenholtz Attorney-Meyer Neishloss et a1.

[5 7 ABSTRACT A process for cultivating an acetic acid-consuming yeastincludes the following steps:

1. An acetic acid-consuming yeast is added to a growth-supportingmixture of aqueous nutrient medium, acetic acid at a low concentrationlevel and gas containing free oxygen.

2. Aerobic culturing of the yeast is initiated with no noticeablestationary growth stage by incrementally introducing small amounts ofacetic acid into the fermentation mass either continuously orintermittently throughout the growth period of the micro-organism.

14 Claims, No Drawings PROCESS FOR CULTIVATING ACETIC ACID-CONTAININGYEASTS This invention relates to a process for cultivating yeastsutilizing a substrate containing acetic acid as a sole source of carbonto promote growth and more particularly to a process wherein propagationof the acetic acid consuming-yeasts is conducted under conditionssubstantially to eliminate the initial stationary phase normallyencountered in the characteristic growth pattern of micro-organisms suchas yeasts.

Critical food shortagesfor both animals and humans in some parts of theworld is a problem of growing concern. The use of fertilizers andimproved farming techniques have greatly increased crop return per acreof cultivated ground. While increased crop yields has resulted in morefood for more people throughout the world, there are still an alarmingnumber of people who are suffering from malnutrition. To alleviatemalnutrition, protein and vitamin foodsupplements have been developedfor both animal and human consumption. Protein concentrates for foodsupplements that are commercially available include fish meal, peanutmeal, cottonseed meaL-soybean meal and micro-organisms such as bacteria,molds, yeasts and the like.

Micro-organisms, because of their high rate of multiplication, havereceived much attention as a source of high quality protein. The abilityof micro-organisms to metabolize carbohydrates is well-known. It isequally well-known, however, that carbohydrates are relatively expensiveraw materials if the desired end product is a relatively inexpensivemicro-organism. Because of the abundance of relatively inexpensive crudeoil deposits, a considerable amount of experimental work has beenconducted utilizing hydrocarbons derived from petroleum as the solesource of carbon for the growth of micro-organisms. A number ofmicro-organisms have been found to grow on petroleum substrates varyingfrom normally gaseous hydrocarbons through normally liquid hydrocarbonsand hydrocarbons that are solid under normal atmospheric conditions.

While hydrocarbons derived from petroleum have been successfullyutilized in supporting the growth of some micro-organisms, particularlyyeasts and bacteria, some difficulty has been experienced in obtaining aprotein product which is completely free from residual hydrocarbons.Various procedures have been developed for obtaining a hydrocarbon-grownprotein product which is free from hydrocarbons but such procedures'haveadversely affected the economics of the overall process.

It is known that some micro-organisms particularly bacteria such asBrevibacterium roseum, Brevibacterium flavum, Brevibacteriumlactofermentus, Corynebacterium acetoacidophilum and Corynebacteriumacetoglutanicum have utilized some organic acids such as citric acid,acetic acid, lactic acid and the like in substrates as the carbon sourcein the fermentative production of L-glutamic acid. These organic acidsbecause of their ready solubility in water cause no problem ofcontamination in recovering fermentation products free from such acids.

While acetic acid has been used as a source of carbon in thefermentation of certain bacteria, acetic acid has not been used as acarbon source in cultivating yeasts because of the low product yieldsobtained due to the inhibitory-growth effect. which acetic acid has onyeasts. In some instances, acetic acid is known to have completelykilled yeasts.

As indicated above, one of the problems which heretofore has beenencountered in cultivating yeasts on an acetic acid-containing substrateis the inhibitorygrowth effect which acetic acid has on the yeasts. Thepoor initial growth response encountered with yeasts cultured on aceticacid, at least in part, is due to the initial stationary phase normallyassociated with the growth of all micro-organisms. During the initialstationary phase, the number of micro-organisms remains constant. Inorder for a fermentation process to be economically attractive, it isdesirable, among other things, to obtain a high concentration ofmicro-organism cells as soon as possible after initiation of thepropagation. Thus, it is desirable to initiate the growth ofmicroorganisms under conditions such that the initial stationary phaseis either eliminated or the time in which the phase is encounteredgreatly reduced.

Typically, micro-organisms grow in the fermentation period in acharacteristic growth pattern which may be designated as follows:

1. Initial stationary phase In this phase, the number of micro-organismsremains constant.

2. Lag phase During this period, the rate of multiplication increaseswith time.

3. Logarithmic growth phase The rate of multiplication remains constant;the generation time is the same throughout the period.

4. Negative growth phase During this phase, the rate of multiplicationdecreases and the average generation time increases. The organismscontinue to increase in number, but at a slower rate than during thelogarithmic phase.

5. Maximum stationary phase The number of living organisms remainsconstant, i.e., the death rate equals the rate of reproduction.

6. Accelerated death phase The number of microorganisms declines withincreasing rapidity. The average rate of death increases to a maximum.

7. Logarithmic death phase In this period, the rate of death isconstant.

In accordance with the prsent invention, a process is provided forcultivating an acetic acid-consuming yeast wherein the propagation ofthe yeast is initiated under conditions such that the initial stationaryphase during which time the number of yeast cells remains constant isgreatly reduced or eliminated.

We have found that the growth of yeast which is cultivated at an activefermentation temperature in the presence of an aqueous nutrient medium,acetic acid and a gas containing free oxygen can be initiated with areduced or no noticeable initial stationary phase only if the aceticacid is present at a low concentration level. According to the presentinvention, an acetic acidconsuming yeast is introduced at a low cellconcentration at an active fermentation temperature at about 20 to about35 C. into a yeast growth-supporting mixture comprising an aqueousnutrient medium, acetic acid, a nitrogen source material and a gascontaining free oxygen. Thereafter, the yeast is cultivated in thepresence of the growth-supporting mixture while additional small amountsof acetic acid and nitrogen source material are incrementally introducedinto the fermentation mass either continuously or intermittentlythroughout a substantial part of the growth period. The amount of theacetic acid and the nitrogen source material introduced at any giventime is such that the total amount of acetic acid and nitrogen presentin the fermentation mass at said time is the optimum amount forachieving, at said time, the maximum rate of growth. The acetic acid andthe nitrogen source material are added to the fermentation mass inincreasing amounts so that the amount of acetic acid and nitrogen addedare about equal to the amount of acetic acid and nitrogen consumed bythe yeast at any given time. If a large amount of either acetic acid orthe nitrogen source material is present at the start of the propagation,the normal initial stationary phase is not substantially reduced.

The acetic acid which is used in the process of the invention can beeither chemically pure acetic acid or commercially available aceticacid, the latter being more economically desirable. In initiating thepropagation of the yeast in accordance with the process of theinvention, a small amount of the acetic acid is added to the nutrientmedium. While the amount of acetic acid added initially, particularlythe maximum amount, may vary slightly depending upon the particularstrain of yeast, we have found that an initial acetic acid concentrationof about 0.05 to about 0.5, preferably about 0.05 to about 0.3 percentby volume based on the volume of the fermentation mass (brew) containingabout 0.2 to about 3 grams of seed yeast (dry) per liter of fermentationmass is sufficient for optimal growth. After propagation of the yeasthas been initiated, additional acetic acid is added continuously orintermittently to the fermentation mass in an amount sufficient tomaintain a reserve acetic acid concentration in the fermentation mass ofabout 0.05 to about 0.5, preferably about 0.05 to about 0.3 percent byvolume of the fermentation mass. The acetic acid can be added eithercontinuously at apprxoimately an exponentially increasing rate orintermittently using approximately exponentially increasing quantities.In any event, the acetic acid is added at a rate about equal to the rateat which it is assimilated by the yeast while simultaneously maintaininga reserve acetic acid concentration in the fermentation mass within therange of about 0.05 to about 0.5 percent (volume/volume). For economicreasons, it is desirable to use the least amount of acetic acidnecessary to initiate and maintain propagation of the yeast thereon. Ifconcentrations of acetic. acid above about 0.5 percent by volume basedon the volume of fermantation mass containing about 0.2 to about 3 gramsof seed yeast (dry) i.e., Pichia membranaefaciens (CS-l90-l) (ATCC20340), per liter of fermentation mass are utilized, there is anundesirable initial stationary phase noted in the propagation. If theseed yeast is Pichia membranaefaciens (ATCC 20101 the maximum aceticacid concentration to avoid an undesirable initial stationary phase inthe propagation is about 0.3 percent (v/v). If more than 0.6 percent(v/v) of acetic acid is employed with Pichia membranaefaciens (CS-l90-l)(ATCC 20340) and if more than 0.4 percent (v/v) of acetic acid isemployed with Pichia membranaefaciens (ATCC 20101), not only is there anundesirable lag in the propagation but also there is a substantialdecrease in yield. For this reason, we prefer to use a small amount ofacetic acid which is sufficient to initiate propagation of the yeastwithout any noticeable lag and which is insufficient to result indecreased yields. Thus, the maximum amount of acetic acid employed mayvary from 0.3 percent with Pichia membranaefaciens (ATCC 20101) to 0.5percent with Pichia membranaefaciens (CS-190-l) (ATCC 20340). We haveobtained optimum yields when the acetic acid is within the range ofabout O.l to about 0.3 percent (v/v). We have noted that the grams ofyeast (dry) grown per gram of acetic acid used in the process of theinvention is virtually independent of the seeding rate.

Yeasts which can be cultivated in the process of the invention are thosewhich normally can assimilate carbon from acetic acid or those whichhave been adapted to assimilate carbon from acetic acid including thoseof the family Cryptococcaceae and especially of the subfamilyCryptococcoideae. Other yeasts which can be employed are those of thefamily Saccharomycetaceae and especially of the sub-familySaccharomycetoideae. A preferred genera of the Cryptoccoideae sub-familyis Candida. A preferred species of the Candida genera is Candida valida.A preferred genera of the Saccharomycetoideae sub-family is Pichia. Apreferred species of the Pichia genera is Pichia membranaefaciens. Ofthese yeasts, a strain of Pichia membranaefaciens is preferred,particularly Pichia membranaefaciens, strain CS-l-l which has beenisolated from soil in which purple grapes are growing. Pichiamembranaefaciens, strain CS-l90-l has been made permanently available bydepositing a culture thereof in the American Type Culture Collection inRockville, Md. This strain has been assigned the ATCC number 20340.

The taxonomic description of Pichia membranaefaciens CS-l90-l (ATCC20340) is as follows:

Pichia mcmbranacfaciens Characteristics CS-l90-l ATCC 20340 GrowthTemperature Response:

30C. Growth 37C. Growth Growth on Malt Extract: Thin, creeping film.slowly formed.

Cell Size: (2.3-4.5) X (3.3-6.0);4. Reproduction: Budding; Rudimentarypseudomycelium; Ascospores; Four, hat-shaped spores per ascus', Asciformed of diploid cells or after conjugation of haploid cells.

Strictly oxidative; No sugar fermentation.

Assimilation of Carbon Compoundsz Glucose Lactose Maltose SucroseGalactose Ethanol L-sorbose Glycerol Lactic Acid Succinic AcidCellobiose Trehalose Melibiosc Melezitose lnulin Soluble Starch D-xyloseL-arabinose D-arabinose D-ribose L-rhamnose Erythritol Adonitol DulcitolMannitol Sorbitol alpha-Methyl-D-glucoside Salicin Gluconoo- -lactoneZ-Keto-gluconatc S-Keto-gluconate Citric Acid Myo-inositol Assimilationof Nitrogen Compounds:

Nitrate Sugar Fermentation:

+(latentl llllllllllllllllllllllll-ll-i Peptone Ammonium sulfate .4.Growth in Vitamin-free Medium: Vitamins Required: Biotin +=Assimilated;-'=Not Assimilated Inasmuch as micro-organisms such as yeasts are madeup of living cells, their growth, as is true with other livingorganisms, depends upon an adequate supply of carbon, hydrogen, oxygen,nitrogen, phosphorus, sulfur and trace amounts of other elementsincluding calcium, potassium, magnesium and iron. Carbon is required forgrowth and energy. Nitrogen is required for synthesis of protein andother nitrogenous materials. Other elements are required for mineralstructure of the cell. In the present process, carbon, hydrogen and someof the oxygen are supplied by the acetic acid; additional oxygen issupplied from the aqueous substrate and from the air, the micro-organismbeing an aerobe; and nitrogen is supplied through the use of ammonium orother nitrogenous inorganic salts. Trace quantities of other elementsnecessary for growth of the yeast may be supplied as impurities in theinorganic salts or these elements may be added directly in extremelysmall amounts. Frequently, sufficient quantities of the trace elementsare present in tap water.

Oxygen, as disclosed hereinabove, is one of the essential elementsrequired to promote the growth of a micro-organism. While pure oxygencan be employed,

we prefer for economic reasons to supply the oxygen as air. In order toeffect an optimum growth of the microorganism, the air should be finelydispersed through the a substrate preferably with agitation at a ratesufficient to form a vortex in the liquid. Depending upon the design ofthe fermentor, various air introducing means can be used includingsingle orifice, half and full ring types with openings for air dischargedirected upwardly and- /or downwardly, and sintered glass percolatortypes with various impellers for lifting air. Impellers, when employed,can be rotated at rates from to 1,000 rpm or more, the particular ratebeing chosen to create vortex in the liquid. Regardless of the type ofair introduction means employed, opertion should 'be such as to avoidexcessive foaming since foaming tends to entrap the micro-organism andremove it from the source of soluble nutrients required in its growth.While the amount of air employed depends somewhat on the size and designof the fermentor, good results are obtained in l4-liter fermentorscontaining 7 liters of brew when employing sterile air at rates of 2 to10 liters per minute.

The nitrogenous materials which can be used in the process of theinvention to supply nitrogen are those compounds of nitrogen which arereadily soluble in water including, by way of example, the ammoniumphosphates, ammonium chloride, ammonium nitrate, ammonium hydroxide,ammonium sulfate and the like. In initiating the propagation of theyeast in accordance with the process of the invention, a small amount ofthe nitrogen source material is added initially either by means of anitrogenous material in the nutrient medium or by separate addition of anitrogenous material to the nutrient medium. While the amount of thenitrogen source material added initially may vary from one material toanother depending upon its nitrogen content, we have found that aninitial nitrogen concentration of about 30 to about 200 mg of nitrogenper liter of fermentation mass containing about 0.2 to about 3 grams ofseed yeast (dry) per liter of fermentation mass is sufficient foroptimal growth. After propagation of the yeast has been initiated,additional nitrogen source material is added continuously orintermittently to the fermentation mass in an amount sufficient tomaintain a reserve nitrogen concentration in the fermentation mass ofabout 30 to about 200 mg of nitrogen per liter of fermentation .mass.The nitrogen source material can be added either continuously atapproximately an exponentially increasing rate or intermittently usingapproximately exponentially increasing quantities. In any event, thenitrogen source material is added at a rate about equal to the rate atwhich it is consumed by the yeast while simultaneously maintaining areserve nitrogen concentration in the fermentation mass of about 30 toabout 200 mg of nitrogen per liter of fermentation mass. For economicreasons, it is desirable to use the least amount of nitrogenous materialnecessary to initiate and maintain propagation of the yeast.

Adequate growth of a micro-organism such as yeast requires not only asource of readily available carbon, hydrogen, oxygen and nitrogen butalso the presence of an aqueous medium containing other nutrients. Themake-up of the aqueous nutrient medium which we employ can vary to someextent depending upon the particular yeast which is used. In general,the nutrient medium comprises a mixture of mineral salts which furnishions of nitrate or nitrite, potassium, ferrous or ferric, calcium,magnesium, phosphate, sulfate, as well as ions of trace elementsincluding zinc, manganese, copper and molybdenum. Ammonium ions tosupply nitrogen have previously been mentioned and are present in theaqueous nutrient medium in small amounts required to give the nitrogenconcentration defined hereinabove. Inasmuch as water is included in thenutrient mixture, many of the mineral salts can be incorporated into thesubstrate in sufficient quantity through the use of tap water. It isdesirable, however, to add the salts to the mixture to insure theirpresence in sufficient quantity for growth of the yeast. The nutrientmixture consists primarily of water, which may constitute about 50 to 99percent by weight or more of the total nutrient mixture. Generally, thewater is employed in an amount normally used in microbial synthesis. Atypical mineral salts medium for the growth of yeasts of the genusPichia, for example Pichia membranaefaciens, in the process of theinvention has the following composition:

Potassium dihydrogen phosphate, KHJO, 2.0 g Magnesium sulfate,MgSO,-7H,O 1.0 g Sodium chloride, NaCl 0.2 g Calcium chloride, CaClyZlhO0.2 g. D-biotin Sag. Boric acid, H l000ug. Copper sulfate, CuSO,-5l-l,0so Potassium iodide, Kl 200 .g Ferric chloride, FeCI,-6H O 400pgManganese sulfate, MnS0,-H,O 800;,tg Sodium molybdate, Na,MoO,-2H,O400;.tg Zinc sulfate, ZnSO '7H,O 800p.g

Distilled water, sufficient to form 1 liter of solution Prior to use inthe process of the invention, ammonium sulfate UNI-[Q 80 is added to thenutrient medium to give a nitrogen concentration of about 30 to about200 mg per liter of nutrient medium.

Another typical mineral salts medium which can be used as a nutrient inthe process of the invention has the following composition:

Potassium dihydrogen phosphate, KH,P0, 1.3 g. Magnesium sulfate, MgSO-7l-l,0 0.2 g.

Distilled water, sufficient to form 1 liter of solution Prior to use inthe process of the invention, diammonium hydrogen phosphate [(NI-I I-IPOis added to the nutrient medium to give a nitrogen concentration ofabout 30 to about 200 mg per liter of nutrient medium.

In growing an acetic acid-consuming yeast on a substrate to which aceticacid and ammonium salts are added, there is a tendency for the pH of theaqueous nutrient medium to drop, i.e., become more acidic. Thus, toprevent a buildup of acidity which adversely affects the growth of theyeast, it is essential to add an alkaline material to restore the pH ofthe aqueous nutrient medium to a desired level. If the pH is notmaintained at a desired level, the growth of the yeast ceases, that iscellular density no longer increases so that a stationary growth phaseis encountered.

The optimum pH of the aqueous nutrient medium depends somewhat upon thenature of the substrate and the particular yeast being cultured. The pHis usually within the range of about 1.5 to about 8. With mineral saltssubstrates, the optimum pH for most yeast cultures is a pH of about 5.When employing a yeast nitrogen base substrate, optimum growth for ayeast of the genus Candida and Pichia, for example, Candida valida andPichia membranaefaciens occurs at a pH of about 2 to 5, a pH of about3.5 being preferred. In order to maintain the pH at any desired level,we may add to the aqueous nutrient medium any suitable alkaline materialsuch as sodium hydroxide, potassium hydroxide, disodium hydrogenphosphate, ammonium hydroxide and ammonia.

The optimum temperature for the growth of the yeast is within the rangeof about to about 35 C. When using a strain of Candida valida or Pichiamembranaefaciens the preferred temperature range is about to about 35 C.

In order to illustrate the improved results obtained in accordance withthe process of the invention, comparative examples are set forthhereinafter. In Examples I to VI and X to XII, the acetic acidconcentration in the brew is maintained within the range of about 0.05to about 0.5 percent (v/v). In Examples VH to IX and XIII to XV theacetic acid concentration in the brew is maintained within the range ofabout 0.5 to about 1.0 percent (v/v).

In the comparative examples, we have utilized Pichia membranaefaciens,strain CS-l90-1 (ATCC 20340) in Examples I to IX and Pichiamembranaefaciens (ATCC 20101) in Examples X to XV. The Pichiamembranaefaciens (ATCC 20340) was isolated through an enrichment cultureprocedure from soil in which grapes were growing in WesternPennsylvania. The yeast was characterized and identified in accordancewith the classification in The Yeasts by J. Lodder and N. J. W.Kreger-Van Rij, North Holland Publishing Co., Amsterdam, 1952. The yeastPichia membranaefaciens (CS-190-1) (ATCC 20340), was found to beidentical in all physiological characteristics with Pichiamembranaefaciens (ATCC 20101) obtained from the American Type CultureCollection, Rockville, Md. Morphologically, Pichia membranaefaciens(CS-190-1) (ATCC 20340) was similar to Pichia membranaefaciens (ATCC20101 differing primarily in cell size. The

cells of the strain of Pichia membranaefaciens (CS-l90-l) (ATCC 20340)were slightly smaller than the cells in Pichia membranaefaciens (ATCC20101). The cells in Pichia membranaefaciens (CS-190-1), on the averagemeasured 6.6;1. 4.5p.. The cells in Pichia membranaefaciens (ATCC 20101on the average measured 7.1 p. 4.9p..

EXAMPLE I Seven liters of an aqueous mineral nutrient medium areintroduced into a 14-liter Pyrex glass fermentor jar equipped with astainless steel head assembly. The head assembly contains ports for theaddition of nutrients and renioval of samples, an agitator shaft, an airsparger line, baffles and a thermometer well. The aqueous nutrientmedium introduced into the fermentor has the following composition basedon one liter of solution:

Potassium dihydrogen phosphate, KH PO, 2.0 g. Magnesium sulfate, MgSO-7H,0 1.0 g. Sodium chloride, NaCl 0.2 g. Calcium chloride, CaCl,-2H,O0.2 g. D-biotin 4p.g. Calcium pantothenate BOOag Folic acid 4 .g.Inositol 4000 13. Niacin 800;,tg. p-aminobenzoic acid 400p.g. Pyridoxinehydrochloride 800 .tg. Riboflavin 400p.g. Thiamine hydrochloride BOOag.Boric acid, H=BO= IOOOug. Copper sulfate, CuSO,-5H,O p.g. Potasiumiodide, KI 200;;g. Ferric chloride, FeCl,-6H,O 400p.g Manganese sulfate,MnSO,-H,O 800p.g Sodium molybdate, Na,MoO -2H,O 400p.g Zinc sulfate,ZnSOflI-LO 800p.g

Distilled water Sufficient to form 1 liter of solution The fermentor isplaced in a water bath which is adjusted to maintain the fermentationmedium at 28 to 30 C. The fermentor is equipped with impellers connectedto a drive mechanism capable of rotating the impellers at rates up to1,000 rpm. The air which is introduced into the fennentor through thesparger is passed through a stainless steel filter tube packed withPyrex glass wool to remove any particles of dust as well as any air-bomemicro-organisms.

Prior to the start of assimilation, 4.62 g. of ammonium sulfate and 7.0ml of acetic acid feedstock which contains 0.533 g. of acetic acid perml are added to the fermentor which contains the seven liters ofnutrient medium. The concentration of acetic acid in the fermentor isthus about 0.05 percent by volume or 50 a1 acetic acid per 100 ml ofnutrient medium. The concentration of nitrogen based on 4.62 g. ofammonium sulfate per 7 liters of nutrient medium is about 140 mg. ofnitrogen per liter of nutrient medium. 4.7 grams of dry yeast of thestrain Pichia membranaefaciens (CS-l-1) (ATCC 20340) is introduced asinoculant into the ferrnentor. The cellular density of the yeast in theferrnentor is thus about 0.67 g. per liter.

Propagation is conducted for 8 hours at 28 to 30 C. with an aerationrate of about 5 liters per minute at impeller speeds of 500 to 700 rpm.During this 8-hour period a total of 54.4 ml of the acetic acidfeedstock (0.533 g. acetic acid/ ml.) and 17.2 ml. of the nitrogensource material is added at hourly intervals in increasing increments.The acetic acid is added to maintain the concentration level at about0.05 percent, i.e., 50 p.l of acetic acid per ml of brew over the 8-hourperiod. Acetic acid concentration levels are determined hourly by gasliquid chromatography (GLC). Aqueous solutions of ammonium sulfate andammonium hydroxide are added to supply nitrogen and to EXAMPLE 11 Theprocedure of Example 1 is repeated using Pichia membranaefaciens(CS-1-190) (ATCC 20340) as the seed yeast except that an acetic acidconcentration of maintain the pH of the fermentation mass within the 50.1 percent is used. A summary of the experimental range of about 3.0 toabout 3.5. The acetic ac1d feeddata and the results obtained in ExampleIi are shown stock and the nitrogen source material are added at the inTable 11.

TABLE rt-sunrniitfOY'EXAMPLE II Nitrogen Agetilc {101g Aceticlaclqsource ee 5 c cone. eve T l Formalin No. additions, additions, found.Volume. 113; 1 111 p11 1 (mg. N/l.) 1 1111. ml. 3 .11.]100 1111. m brew,g.

3.4 0.03 (130, 2) 0.0 0.0 115 3.4 0. 03 130.2) 0.0 2. 0 62 71 3.3 0.03(130.2) 1.0 3.2 7, 003.0 5.11 3.3 0. 03 (130.2) 1.2 3.7 72 7, 012.0 0.03.2 0.01 127.4) 1.3 4.0 80 7,010.2 7.7 3. 2 0. 01 (127. 4) 2. 4 7. 5 807, 028.1 J. 2 3.2 0.33 (123.2) 2.0 0.1 7,040.1 11.1 3.2 0. 02 123.3) 4.414.1 78 7,0511. 0 13.7 3.2 0.117 (121.8) .1.1 13.1 55 7,075.8 10.0 Total1s. 2 1 s7. 0

1 Determination made at the end of each hour. 1 Feed additions are madeat the beginning of each hour.

1 The feedstock acetic 4 Conditions at start:

beginning of @5611 hour '11;'aiaamisa sfissfinetar the anticipated yeastgrowth. Thus, the concentration of 2 tion period the cellular density isabout 15.5 g. of dry matter per seven liters, or about 2.2 g. per liter.

A summary of the experimental data and the results obtained in Example 1are shown in Table I.

acid contains 0.533 g. acetic acid/1111.

4.7 g. of seed yeast; 7-L of nutrient medium; 4.62

of feedstock acetic acid (0.1% acetic acid or 100 d. accticacid/1001111.).

5 As (NI-101501solutioneontaining 40.00 111 f40s7NI-I10H solutioncontaining 52.50 mg. i1 g.

g. of (311102801; and 14.0 1111. (7.5 g.)

EXAMPLE III The procedure of Example I is repeated using Pichiamembranaefaciens (CS-1904) (ATCC 20340) as the Nitrogen Acetic acidAcetic acid source feedstock cone. level Total yeast Formalin No.additions. additions. found, Volume, (dry) in pH 1 (mg. N/l.) l 1111.1111. J .11.]100 111l. 1n1. brew, 1:.

Time, hrs

Total 17. 2

1 Determination made at the end of each hour. 1 Feed additions are madeat the beginning of each hour. 3 The feedstock acetic acid contains0.533 g. acetic acid/m1.

4 Conditions at start: 4.7 g. of seed yeast; 7L of nutrient medium; 4.62g. of (NH1)7S04; and 7.0 ml. (3.7 g.)

of feedstock acetic acid (0.50% acetic acid or 50 .11. acetic acid/100n1l.).

5 As (NAmSOt solution containing 40.00 mg. N/ml. 5 As NHtOH solutioncontaining 52.50 mg. N /1n1. 7 20.0 g.

seed yeast except that an acetic-acid concentration of 0.2 percent isused. A summary of the experimental data and the results obtained inExample 111 are shown in Table III.

Nitrogen Acetic acid Acetic acid source feedstock cone. level Totalyeast Fornntlin No. additions, additions. found. Volume, (dry) in p11(111g. NH.) 1 1nl. ml. 3 11/100 n11. n11. brew,

Time, hrs: 0... 3.3 0.03 (130.2) 0.0 0.0 4.1 1 3.2 0. 03 (130. 2) 5 0.it 2. i) 5. 2 2 3.2 0.03 (130.2) 1.0 3.2 5.3 3 3. 2 0. 03 (130. 2) 1. 23. 7 t1 4 3.2 0.03 (130.2 1.3 4.0 1.3 5 3. 2 0.00 (126.0 2. 4 7. 5 0. 06 3.2 0. (124.6 6 2.0 0.1 10.8 7 3. 2 0. 00 (120. 0 t 4. 4 14.1 13. 3 83. 2 0. 811 (124. 6) 0 4. 7 15.0 16. 2

Total 1s 8 1 50. 5

t The feedstock acetic acid conttins 0.533 g. acetic acid/ml.

'-' Feed additions are made at the beginning of each hour.

I Determination made at the end of each hour.

1 Conditions at start: 4.7 a. oi seed yeast; FL 01 nutrient medium; 4.62g. oi (N111)2S01; and 23.0 1111. (14.0 g.) of feedstock acetic acid(0.1.7 acetic acid or 200 11. acetic acid/ 1111.).

EXAMPLE v The procedure of Example I is repeated using Pichiamembranaefaciens (CS-l90-1) (ATCC 20340) as the seed yeast except thatan acetic acid concentration of 0.4 percent is used. A summary of theexperimental data and the results obtained in Example V are shown inTable V.

EXAMPLE IV The procedure of Example 1 is repeated using Pichiamembranaefaciens (CS-190-l) (ATCC 20340) as the TABLE V.SUMMARY OFEXAMPLE V Nitrogen Acetic acid Acetic acid source feedstock cone. levelTotal yeast Formalin No. additions. additions, found, Volume, (dry) inp11 1 (mg. N/l.) 1 m1. ml. 3 p1./100 ml. ml.

726538031 a -m5 07 3 0 3 0 11l OMWMMOMMW 1 Determination made at the andof each hour.

1 Feed additions are made at the beginning of each hour.

3 The feedstock acetic acid contains 0. 533 g. acetic acid/ml.

4 Conditions at start: 4. 7 g. of seed yeast; 7-L of nutrient medium; 4.62 g. of (NIIQZSO; and 56. 0 ml. (214. is g.) of feedstock acetic acid(0.4% acetic acid or 400 p1. acetic acid/100 ml.).

6 As (NH4)2SO4 solution containing 49. 00 mg. N lml.

f 305 51114011 solution containing 52. 50 g. N/mi.

seed yeast except that an acetic acid concentration of It will be notedfrom the data in Table V that the total yeast in the brew increased from4.7 to 16.1 grams, an

0.3 percent is used. A summary of the experimental data and the resultsobtained in Example IV are shown in Table IV.

TABLE IV.SUMMARY OF EXAMPLE IV 15 5 6 7 HHLQMGW 111 0800211 0 RMZ 0oimfimww 000000000 090234941 LLLZZ15 50506 00 Time, hrs.:

Total..

1 Determination made at the end of each hour.

2 Feed additions are made at the beginning of each hour. 3 The feedstockacetic acid contains 0.533 g. acetic acid/ml. 4 Conditions at start 4.7g. of seed yeast; 7-L of nutrient medium; 4.62 g. of (N 119 80 and 42.0mi. (22.4 g.) of feedstock acetic acid (0.3% acetic acid or 300 1.acetic acid/100 ml.).

5 As (NH|)2SO4 solution containing 49.00 mg. N lml. psszliHioH solutioncontaining 52.50 mg. N/ml.

lt will be noted from the data in Table IV that the total yeast in thebrew increased from 4.7 to 16.2

EXAMPLE VI grams, an increase of 11.5 grams (71.0 percent yield/8 Theprocedure of Example 1 is repeated using Pichia hrs.) and thatpropagation was initiated without noticemembranaefaciens (CS-1904) (ATCC20340) as the able lag.

seed yeast except that an acetic acid concentration of 0.5 percent isused. A summary of the experimental It will be noted from the data inTable VII that the data and the results obtained in Example VI are showntotal yeast in the brew increased from 4.6 to 14.0 in Table VI.

grams, an increase of 9.4 grams (67.1 percent yield/8 TABLE IL-SUMMARY FEXAMPLE VI Nitrogen Acetic acid Acetic acid source feedstock cone. levelFormalin No. additions, additions,

Total yeast found, Volume, (dry) in ml. 3 l./100 mlJ ml. brow, g.

ma amm a 002705101 0 9mawam4 lomnwam TotaL.. 54.4

1 Determination made at the end of each hour.

1 Feed additions are made at the beginning of each hour.

3 The feedstock acetic acid contains 0.533 g. acetic acid/ml.

4 Conditions at start: 4.7 g. of seed yeast; 7-L oi nutrient medium;4.02 g. of (N111) :SO 1; and 70.0 ml of feedstock acetic acid (0. 5%acidic acid or 500 pl. acetic acid/100 1111.).

5 As (NHmSOt solution containing 49.00 mg. N lml.

As NI-ii ()11 solution containing 52.50 mg. N/ml. 7 20. 0 g.

It will be noted from the data in Tabie VI that the total yeast in thebrew increasd from 4.7 to 15.4 grams,

hrs.) but that there was a lag in the initiation of the propagation.

an increase of 10.7 grams (69.5 percent yield/8 hrs.) and thatpropagation was initiated without noticeable lag.

EXAMPLE VIII EXAMPLE VII The procedure of Example I is repeated usingPichia membranaefaciens (CS-l-l) (ATCC 20340) as the The procedure ofExample I is repeated using Pichia membranaefaciens (CS-190-1) (ATCC20340) as the seed yeast except that an acetic acid concentration of 0.8percent is used. A summary of the experimental data and the resultsobtained in Example VIII are shown in Table VIII.

"TA'ia'Ln VILQSUMMARY OFEXAMPIIE vn Nitrogen Acetic acid Acetic acidsource feedstock conc. level Total yeast Formalin No. additions,additions, found, Volume, (dry) in pH 1 (mg. N/l.) 1 ml. mi 1 3 ,tL/IOOm1. n11. brew, g

Time, hrs:

090050005 0 0L0 L2 L44 5.0 66666 5478191 w o os m u 0 0L00 Ln .0 000000900 TotaL......

1 Determination made at the end of each hour.

l. acetic acid/ ml.). 5 As (NH4)zSO4 solution containing 39.14 mg. N/ml. 2 12k; 5*IH4OH solution containing 53.62 mg. N/ml.

2 Feed additions are made at the beginning of each hour.

3 The feedstock acetic acid contains 0.536 g. acetic acid/ml.

4 Conditions at start: 4.6 g. of seed yeast; 7-L of nutrient medium;4.62 g. of (NH4)1SO4; and 84.0 ml. (45.0 g.) of feedstock acetic acid(0.6% acetic acid or 600 t TABLE Vfiil 's'U'MMARYoF EXAMPLE Vin000000087 d d d d n xmxmxmaw 000000000 0 4 A A AmA AmA 00 000000 00000000 0 0 0 0 LLL0 0 Time, hrs.:

1 Determination made at the end of each hour.

Feed additions are made at the beginning of each hour. 3 The feedstockacetic acid contains 0.536 g. acetic acid/ml. 4 Conditions at start' 4.6g. of seed yeast; 7-L of nutrient medium; 4.62 g. of (NHmSO and 112.0ml. (60.0 g.) of feedstock acetic acid (0.8% acetic acid or 800 l.acetic acid/100 ml.).

2 As (NIIOESOI solution containing 40.14 mg. N/nil.

TABLE XI X XI XII XIII XIV ExampleNumber 4066008 000053 3 33 201192 0 550 6 1.3529 mod l Acetic acid, pcreent....

In medium initially, g. Added, g. Total in medium, g. Unused, Used, g

Yeast (dry):

EXAMPLE IX The procedure of Example I is repeated using Pichiamembranaefaciens (CS-l90-l) (ATCC 20340) as the 000.0 0w. 3 3 Y. 0.00.0O 30 10- OVA No \in Gross yield, g.. seed yeast except that an aceticacid concentration of 21-hour yield, e en mm. o 1.0 percent 18 used. Asummary of the experimental 10 ,.,,,f, ,,,,,;Q.j.,5}3,,,.,, data d h ultobtained in Example IX are shown in Table IX.

'Iotnl yeast (dry) in mi. brew, g.

l Determined in steam distillation.

TABLE IX.--SUMMARY OF EXAMPLE 1X Nitrogen Acetic acid Acetic acid sonreefeedstock cone. level Formalin No. additions. additions. found. Volume,pH 1 (mg. N/l.) l mlfl ml. 3 H100 nil.

Time, hrs.:

O 0 0 .HI LLLL It will be noted from the comparative data in Table Ithat good yields of yeast were obtained with acetic acid concentrationsbelow 0.6 percent. However, when the substrate contained from 0.4 to 1.0percent of acetic acid there was a noticeable lag in the propagation.For this reason, the range of acetic acid concentration when usingPichia membranaefaciens (ATCC 20101) preferably does not exceed 0.3percent. Optimum results were obtained with an acetic acid concentrationof about 0.05 to about 0.2 percent.

VI VII VIII III IV .000 41. acetic acid/1001111.).

It will be noted from the data in Table IX that the growth of yeast wascompletely inhibited, there being X TABLE x Total. 1 Determination madeat the end of each hour.

' Conditions at start: 4.0 g. of seed yeast; 7-L ofnutricnt medium; 4.62g. of (NIIdzSOi; and 140.0 ml. (75.0 g.)

In order to present for ready comparison the affect of utilizing from0.05 to 1.0 percent of acetic acid in no more yeast at the end of eighthours than there was at the beginning of the fermentation.

the substrate as a carbon source in cultivating Pichia membranaefaciens(CS-I90-l) (ATCC 20340), the results obtained in Examples I to IX aresummarized in T b e X- 1 The feedstock acetic acid contains 0.530 g.acetic acid/ml. 1 Feed additions are made at the beginning of each hour.

of feedstock acetic acid (1.0% acetic acid or 1 5 As (NIIOzSO; solutioncontaining 40.14 mg. N/ml. I 1.7

Example number" 602266 Dim-1 120 4 2733 %m50.3 344 500. Gone 5 00 Aceticacid. percent... In mcdimn initial] Seed, z.

Net. yield. g..I Gross yield, g...... ti-hour yield. perccn When theprocess of the invention is operated in a continuous manner, a fractioncontaining the desired yeast product is continuously removed from thefermentation mass. The desired yeast product is then separated from thewithdrawn fraction by conventional means such as by centrifuging.Thereafter, the yeast is washed one to three times with tap water andfinally dried under conditions sufficiently mild to avoid autoiysis butunder conditions sufi'lciently severe to assure recovery of a nonviableyeast product containing not more than about 10 percent moisture,usually about 3 to about 5 percent moisture. The drying temperature toinsure the recovery of nonviable cells in an oven is within the range ofabout 50 to about (3. if spray drying is employed, the temperature ofthe dryer may be in the order of about 65 C. without adversely affectingthe quality of the yeast. In drum drying, a higher drying temperature ofabout C. can be employed.

The make-up of Pichia membranaefaciens (CS-- EXAMPLES X TO XV Theprocedure of Example I is repeated except that Iielu'urrmnhranurfiuricns (ATCC 20 I (ll is used as the instead of (CS-l90-l)(ATCC 20340). The substrate in these ex- G rams of yeast grown aceticacid used Lug in PIODZIZHIIOIL.

iDeterniincd by steam distillation. g w I It will be noted from thecomparative data in Table X that good yields of yeast were obtained withacetic acid concentrations of 0.05 to 0.6 percent. However, when thesubstrate contained more than 0.5 percent of acetic acid there was anoticeable lag in the propagation. for this reason, the range of aceticacid concentration when using Pichia membranaefaciens (CS-l90-1) (ATCC20340) preferably does not exceed 0.5 percent. Optimum results wereobtained with an acid concentration of about 0.1 to about 0.4 percent.

seed yeast amples contained 0.05, 0.2, 0.4, 0.6, 0.8 and 1.0 percent ofacetic acid. The results obtained in these examples are summarized inTable Xi.

1) (ATCC 20340) and (ATCC 20101) obtained in the process of the presentinvention may vary slightly from one fermentaion to another. A typicalchemical composition of the dried product is as follows:

Pichia membranaefaciens Percent by Weight ATCC 20340 ATCC 20101" Totalnitrogen 7.49 7.92 Crude protein 46.81 49.50 Total Amino acids 36.8040.62

" Grown on a reserve acetic acid concentration of 0.1%

" Grown on a reserve acetic acid concentration of 0.2%

A summary of the amino acid profile fortypical Pichia membranaefaciensATCC 20340 and ATCC 20101 grown on reserve acetic acid concentrations of0.1 and 0.2 percent, respectively, are as follows:

Pichia membranaefaciens,

Percent by weight of Dried Yeast Product Amino Acids ATCC 20340 ATCC20101" Lysine 2.88 3.25 l-listidine 0.93 0.95 Arginine 3.34 3.36Threonine 2.1 l 2.22 Valine 2.22 2.82 Methionine 0.66 0.69 lsoleucine1.91 2.17 Leucine 2.99 3.53 Phenylalanine 1.74 2.15 Tryptophan 0.87 0.73Aspartic Acid 3.59 4.00 Serine 1.76 2.16 Glutamic Acid 4.54 4.38 Proline1.13 1.40 Glycine 1.75 2.16 Alanine 2.31 2.93 Tyrosine 1.65 1.32 Cystine0.45 0.41

' Grown on a reserve acetic acid concentration of 0.1% Grown on areserve acetic acid concentration of 0.2

While our invention has been described with reference to variousspecific examples and embodiments, it will be understood that theinvention is not limited to such examples and embodiments and may bevariously practiced within the scope of the claims hereinafter made.

We claim:

1. A process for cultivating an acetic acid-consuming yeast whichcomprises introducing an acetic acidconsuming yeast at an activefermentation temperature of about to about 35 C. into a yeastgrowthsupporting mixture comprising an aqueous nutrient medium, aceticacid, a nitrogen source material and a gas containing free oxygen, saidmixture initially containing about 0.05 to about 0.5 volume percent ofacetic acid and about 30 to about 200 mg. of nitrogen per liter offermentation mass and about 0.2 to about 3 grams of yeast per liter offermentation mass and cultivating the acetic acid-consuming yeast insaid mixture while incrementally introducing into said mixtureadditional acetic acid and nitrogen source material throughout thegrowth period of the yeast, the amount of the acetic acid and thenitrogen source material introduced at any given time being such thatthe total amount of acid present in said mixture at said time is about0.05 to about 0.5 percent by volume and the total amount of nitrogenpresent in said mixture at said time is about 30 to about 200 mg. ofnitrogen per liter of fermentation mass.

2. The process of claim 1 wherein the yeast is of the familySaccharomycetaceae.

3. The process of claim 2 wherein the yeast is of the subfamilySaccharomycetoideae.

4. The process of claim 3 wherein the yeast is of the genus Pichia.

5. The process of claim 4 wherein the yeast is of the species Pichiamembranaefaciens.

6. The process of claim 4 wherein the yeast is of the strain Pichiamembranaefaciens, (ATCC 20340).

7. The process of claim 5 wherein the pH of the aqueous nutrient mediumis within the range of about 2 to about 5.

8. The process of claim 1 wherein the oxygencontaining gas is air.

9. A process for cultivating an acetic acid-consuming yeast of the genusPichia which comprises introducing the yeast at a temperature of about25 to about 35 C. into a yeast growth-supporting mixture comprising anaqueous nutrient medium whose pH is maintained within the range of about2 to about 5, acetic acid, a nitrogen source material and air, saidmixture initially containing about 0.05 to about 0.5 percent by volumeof acetic acid, about 30 to about 200 mg. of nitrogen per liter offermentation mass and about 0.2 to about 3 grams of yeast per liter offermentation mass and cultivating the yeast in said mixture whileincrementally introducing into said mixture additional acetic acid andnitrogen source material througout the growth period of the yeast, theamount of the acetic acid and the nitrogen source material introduced atany given time being such that the total amount of acetic acid presentin said mixture at said time is about 0.05 to about 0.5 percent byvolume and the total amount of nitrogen present in said mixture at saidtime is about 30 to about 200 mg of nitrogen per liter of fermentationmass.

10. The process of claim 9 wherein the yeast is of the species Pichiamembranaefaciens.

11. A process for cultivating an acetic acidconsuming yeast of the genusPichia which comprises introducing the yeast at a temperature of about25 to about 35 C. into a yeast growth-supporting mixture comprising anaqueous nutrient medium whose pH is maintained within the range of about2 to about 5, acetic acid, a nitrogen source material and air, saidmixture initially containing about 0.05 to about 0.3 percent by volumeof acetic acid, about 30 to about 200 mg of nitrogen per liter offermentation mass and about 0.2 to about 3 grams of yeast per liter offermentation mass; cultivating the yeast in said mixture whileincrementally introducing into said mixture additional acetic acid andnitrogen source material throughout the growth period of the yeast, theamount of the acetic acid and the nitrogen source material introduced atany given time being such that the total amount of acetic acid presentin said mixture at said time is about 0.05 to about 0.3 percent byvolume and the total amount of nitrogen present in said mixture at saidtime is about 30 to about 200 mg of nitrogen per liter of fermentationmass; separating a yeast fraction from the fermenthe strain Pichiamembranaefaciens (ATCC 20101 UNITED STATES PATENT OFFICE CERTIFICATE OFCORRECTION Patent No. 3I775'252 Dated November 27, 1973 V Inventofla)Norma A. Kinsel and William W. Leathen It is certified that errorappears in the above-identified patent and that said Letters Patent arehereby corrected as shown below:

Column 1, line 56, "acetoglutanicum" should read acetoglutamicum Column3, line 35, "apprxoimately" should read I approximately Column 5, line39, after "create" and before "vortex" insert Column 10, Table III,

Total Yeast Total Yeast (Dry) in (Dry) in Brew, g. Brew, g.

should read 4.7 4.7 5.2 5 .2 5.8 5 .8 5 .6 6 .5

Table III, Footnote 1 should read Determination made at the end of eachhour .-7

Table III, Footnote 3 should read The feedstock acetic acid contains0.533 g. acetic acid/ml Column 11, Table IV, in the footnote insert 6before "As NH OH" UNITED STATES PATENT OFFICE I CERTIFICATE OFCORRECTION Patent No, 3,775,252 D ted November 27, 1973 Inventor) NormaA. Kinsel and'William W. Leathen It is certified that error appears inthe above-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 13, Table VI, Footnote 4, after "70.0 ml" and before "of" insert(37.3 g.)

Column 13, Table VII, Footnote 5, "39.14 mg." should read Column 15,line 56 "for" should read For Column 18, line 9, "The process of claim5" should read The process of claim 1 line 27, "througout" should readthroughout Signed and sealed this 23rd day of July 1.?7Lp.

(SEAL) Attest:

MCCOY M. GIBSON, JR. I C. MARSHALL DANN Attesting Officer Commissionerof Patents

1. AN ACETIC ACID-CONSUMING YEAST IS ADDED TO A GROWTH-SUPPORTINGMIXTURE OF AQUEOUS NUTRIENT MEDIUM, ACETIC ACID AT A LOW CONCENTRATIONLEVEL AND GAS CONTAINING FREE OXYGEN.
 2. AEROBIC CULTURING OF THE YEASTIS INITIATED WITH NO NOTICEABLE STATIONARY GRWOTH STAGE BY INCREMENTALLYINTRODUCING SMALL AMOUNTS OF ACETIC ACID INTO THE FERMENTATION MASSEITHER CONTINUOUSLY OR INTERMITTENTLY THROUGHOUT THE GROWTH PERIOD OFTHE MICRO-ORGANISM.
 2. The process of claim 1 wherein the yeast is ofthe family Saccharomycetaceae.
 3. The process of claim 2 wherein theyeast is of the subfamily Saccharomycetoideae.
 4. The process of claim 3wherein the yeast is of the genus Pichia.
 5. The process of claim 4wherein the yeast is of the species Pichia membranaefaciens.
 6. Theprocess of claim 4 wherein the yeast is of the strain Pichiamembranaefaciens, (ATCC 20340).
 7. The process of claim 5 wherein the pHof the aqueous nutrient medium is within the range of about 2 to about5.
 8. The process of claim 1 wherein the oxygen-containing gas is air.9. A process for cultivating an acetic acid-consuming yeast of the genusPichia which comprises introducing the yeast at a temperature of about25* to about 35* C. into a yeast growth-supporting mixture comprising anaqueous nutrient medium whose pH is maintained within the range of about2 to about 5, acetic acid, a nitrogen source material and air, saidmixture initially containing about 0.05 to about 0.5 percent by volumeof acetic acid, about 30 to about 200 mg. of nitrogen per liter offermentation mass and about 0.2 to about 3 grams of yeast per liter offermentation mass and cultivating the yeast in said mixture whileincrementally introducing into said mixture additional acetic acid andnitrogen source material througout the growth period of the yeast, theamount of the acetic acid and the nitrogen source material introduced atany given time being such that the total amount of acetic acid presentin said mixture at said time is about 0.05 to about 0.5 percent byvolume and the total amount of nitrogen present in said mixture at saidtime is about 30 to about 200 mg of nitrogen per liter of fermentationmass.
 10. The process of claim 9 wherein the yeast is of the speciesPichia membranaefaciens.
 11. A process for cultivating an aceticacid-consuming yeast of the genus Pichia which comprises introducing theyeast at a temperature of about 25* to about 35* C. into a yeastgrowth-supporting mixture comprising an aqueous nutrient medium whose pHis maintained within the range of about 2 to about 5, acetic acid, anitrogen source material and air, said mixture initially containingabout 0.05 to about 0.3 percent by volume of acetic acid, about 30 toabout 200 mg of nitrogen per liter of fermentation mass and about 0.2 toabout 3 grams of yeast per liter of fermentation mass; cultivating theyeast in said mixture while incrementally introducing into said mixtureadditional acetic acid and nitrogen source material throughout thegrowth period of the yeast, the amount of the acetic acid and thenitrogen source material introduced at any given time being such thatthe total amount of acetic acid present in said mixture at said time isabout 0.05 to about 0.3 percent by volume and the total amount ofnitrogen present in said mixture at said time is about 30 to about 200mg of nitrogen per liter of fermentation mass; separating a yeastfraction from the fermentation mass; and recovering yeast from saidfraction.
 12. The process of claim 11 wherein the yeast is of thespecies Pichia membranaefaciens.
 13. The process of claim 11 wherein theyeast is of the strain Pichia membranaefaciens, (ATCC 20340).
 14. Theprocess of claim 11 wherein the yeast is of the strain Pichiamembranaefaciens (ATCC 20101).